LAAO-<i>Bl</i> induces necrosis and apoptosis in renal epithelial cell lines.

<p>(A) MDCK (a) and HK-2 (b) cells were treated with LAAO-<i>Bl</i> (1.56–50/100 μg/mL) for 12 h. After treatment, cell viability was determined by MTT. (B) Percentage release of the lactate dehydrogenase enzyme of MDCK (a) and HK-2 (b) cells treated with the indicated concentrations of LAAO-<i>Bl</i>. Triton (7%) was used as positive control. (C) Annexin V⁺/PI^-, Annexin V⁺/PI⁺ and AnnexinV^-/PI⁺-populations were analyzed by flow cytometry in MDCK (a) and HK-2 (b) cells for 12 h after treatment with LAAO-<i>Bl</i>. Staurosporine (1 μg/mL) and doxorubicin (10 μM) for 12h were used as positive controls. All data are representative of three independent experiments in triplicate and are expressed as mean ± S.E.M. *Significantly different from control group (p<0.05, one way ANOVA, Dunnett post-test).</p

Transcriptome Analysis in Venom Gland of the Predatory Giant Ant <i>Dinoponera quadriceps</i>: Insights into the Polypeptide Toxin Arsenal of Hymenopterans

<div><p>Background</p><p><i>Dinoponera quadriceps</i> is a predatory giant ant that inhabits the Neotropical region and subdues its prey (insects) with stings that deliver a toxic cocktail of molecules. Human accidents occasionally occur and cause local pain and systemic symptoms. A comprehensive study of the <i>D. quadriceps</i> venom gland transcriptome is required to advance our knowledge about the toxin repertoire of the giant ant venom and to understand the physiopathological basis of Hymenoptera envenomation.</p><p>Results</p><p>We conducted a transcriptome analysis of a cDNA library from the <i>D. quadriceps</i> venom gland with Sanger sequencing in combination with whole-transcriptome shotgun deep sequencing. From the cDNA library, a total of 420 independent clones were analyzed. Although the proportion of dinoponeratoxin isoform precursors was high, the first giant ant venom inhibitor cysteine-knot (ICK) toxin was found. The deep next generation sequencing yielded a total of 2,514,767 raw reads that were assembled into 18,546 contigs. A BLAST search of the assembled contigs against non-redundant and Swiss-Prot databases showed that 6,463 contigs corresponded to BLASTx hits and indicated an interesting diversity of transcripts related to venom gene expression. The majority of these venom-related sequences code for a major polypeptide core, which comprises venom allergens, lethal-like proteins and esterases, and a minor peptide framework composed of inter-specific structurally conserved cysteine-rich toxins. Both the cDNA library and deep sequencing yielded large proportions of contigs that showed no similarities with known sequences.</p><p>Conclusions</p><p>To our knowledge, this is the first report of the venom gland transcriptome of the New World giant ant <i>D. quadriceps</i>. The glandular venom system was dissected, and the toxin arsenal was revealed; this process brought to light novel sequences that included an ICK-folded toxins, allergen proteins, esterases (phospholipases and carboxylesterases), and lethal-like toxins. These findings contribute to the understanding of the ecology, behavior and venomics of hymenopterans.</p></div

LAAO-<i>Bl</i> activates Bax, caspases 3 and 7 in epithelial kidney cells.

<p>(A) Caspase-3/7 activity was determined in the presence of the fluorogenic Ac-DEVD-afc substrate after treatment with LAAO-<i>Bl</i> (12 h) in the presence or absence of zVAD (10 μM) in MDCK (a) and HK-2 cells (b). (B-C) Western blot analyses were performed to detect the effects of LAAO-<i>Bl</i> on changes in Caspase-3 (a), -7 (b) and Bax (c) expressions from total protein of MDCK (B) and Caspase-3 (a), -7 (b) HK-2 (C) cell extracts. Percentage of Caspase-3 (a), -7 (b) and Bax (c) expression in MDCK (B) and caspase-3 (a) and caspase-7 (b) HK2 (C) treated with LAAO-<i>Bl</i> for 12 h. Data normalized in relation to Control group. Protein bands were analyzed and normalized to the densitometric values of α-tubulin. Doxorubicin was used as positive control (10 μM). Data are representative results of three independent experiments.</p

Summary of assembled contigs derived from RNA sequencing and comparison with the ESTs of the <i>D. quadriceps</i> venom gland transcriptome.

<p>Summary of assembled contigs derived from RNA sequencing and comparison with the ESTs of the <i>D. quadriceps</i> venom gland transcriptome.</p

Distribution of the Hymenoptera species as determined by best protein hits.

<p>The percentages of homologs from distinct hymenopterans species with which the 6,429 contigs of the <i>D. quadriceps</i> venom gland shared the high sequence similarities.</p

Multiple alignments of deduced amino acid sequences of different identified dinoponeratoxins from the <i>D. quadriceps</i> venom gland transcriptome and <i>D. australis</i>.

<p>Deduced <i>D. quadriceps</i> dinoponeratoxin cDNA precursor sequences (contig_1 and contig_9). RNA deep sequencing contigs (contig_2 and contig_145) were compared to mature peptide sequences from <i>D. australis</i> (Da-3177 and Da-3105) (part A) and from another species of <i>Dinoponera</i> (Da-2501) (part B). ClustalW was used to multi-align the sequences. Identical amino acid residues are marked with asterisks. Stretches of deduced amino acid sequences supported by EST sequences are boxed. Signal peptides (pre-peptides) are doubled underlined, and pro-regions of pro-peptides are shown with a single line under the sequence. Contigs 1 and 9 were first identified in the EST library, and contigs 2 and 145 came primarily from RNA-Seq.</p

Dinoponera quadriceps in the field and its dissected venom apparatus.

<p>Part A - A single specimen of D. quadriceps protecting the nest's entrance. Part B - Dissected <i>D. quadriceps</i> venom apparatus (×40). Abbreviations: <i>Dg</i> - Doffur's gland; <i>cv</i> - convoluted gland (not observable); <i>st</i> - secretory tubule; <i>vs</i>- venom sac; sg – sting.</p

General overview of the transcripts in the <i>D. quadriceps</i> venom gland identified by deep RNA sequencing.

<p>Annotation of contigs from the RNA-Seq assembly of the giant ant venom following the conventions of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087556#pone-0087556-g002" target="_blank">figure 2</a>; i.e., the E-value cut-off was at least 1.0E-5 for BLASTX functional comparison.</p

Multiple alignments of the deduced amino acid sequences of venom allergen I (Sol i 1) from <i>D. quadriceps</i> with known sequences from different ant and wasp species.

<p>The identical and conserved amino acid residues of diverse ant and wasp hymenopterans are highlighted in black and gray, respectively. Dots represent gaps.</p

LAAO-<i>Bl</i> induces necrosis and apoptosis in renal epithelial cell lines.

<p>(A) MDCK (a) and HK-2 (b) cells were treated with LAAO-<i>Bl</i> (1.56–50/100 μg/mL) for 12 h. After treatment, cell viability was determined by MTT. (B) Percentage release of the lactate dehydrogenase enzyme of MDCK (a) and HK-2 (b) cells treated with the indicated concentrations of LAAO-<i>Bl</i>. Triton (7%) was used as positive control. (C) Annexin V⁺/PI^-, Annexin V⁺/PI⁺ and AnnexinV^-/PI⁺-populations were analyzed by flow cytometry in MDCK (a) and HK-2 (b) cells for 12 h after treatment with LAAO-<i>Bl</i>. Staurosporine (1 μg/mL) and doxorubicin (10 μM) for 12h were used as positive controls. All data are representative of three independent experiments in triplicate and are expressed as mean ± S.E.M. *Significantly different from control group (p<0.05, one way ANOVA, Dunnett post-test).</p